1. Field of Invention
The present invention relates to certain 2-indolinone compounds which modulate the activity of protein kinases (xe2x80x9cPKsxe2x80x9d) and phosphatases. The compounds of this invention are therefore useful in treating disorders related to abnormal PK activity. Pharmaceutical compositions comprising these compounds, methods of treating diseases utilizing pharmaceutical compositions comprising these compounds and methods of preparing them are also disclosed.
2. State of the Art
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (xe2x80x94OH) of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases and protein phosphatases are generally divided into two groupsxe2x80x94receptor and non-receptor type proteins. Most receptor-type protein tyrosine phosphatases contain two conserved catalytic domains, each of which encompasses a segment of 240 amino acid residues. Saito et al., 1991, Cell Growth and Diff. 2:59-65. Receptor protein tyrosine phosphatases can be subclassified further based upon the amino acid sequence diversity of their extracellular domains. Saito et al, supra; Krueger et al., 1992, Proc. Natl. Acad. Sci. USA 89:7417-7421.
Protein kinases and protein phosphatases are also typically divided into three classes based upon the amino acids they act upon. Some catalyze the addition or hydrolysis of phosphate on serine or threonine only, some catalyze the addition or hydrolysis of phosphate on tyrosine only, and some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
Tyrosine kinases can regulate the catalytic activity of other protein kinases involved in cell proliferation. Protein kinases with inappropriate activity are also involved in some types of cancer. Abnormally elevated levels of cell proliferation are associated with receptor and non-receptor protein kinases with unregulated activity.
In addition to their role in cellular proliferation, protein kinases are thought to be involved in cellular differentiation processes. Cell differentiation occurs in some cells upon nerve growth factor (NGF) or epidermal growth factor (EGF) stimulation. Cellular differentiation is characterized by rapid membrane ruffling, cell flattening, and increases in cell adhesion. Chao, 1992, Cell 68:995-997.
In an effort to discover novel treatments for cancer and other diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Some small organic molecules form a class of compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases are bis-monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992), styryl compounds (by Levitzki, et al., U.S. Pat. No. 5,217,999, and entitled xe2x80x9cStyryl Compounds which Inhibit EGF Receptor Protein Tyrosine Kinase), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO 91/15495).
The compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous therapeutics as they can become highly bioavailable after being administered orally to patients. However, many of these protein kinase inhibitors only weakly inhibit the function of protein kinases. In addition, many inhibit a variety of protein kinases and will therefore cause multiple side-effects as therapeutics for diseases.
Despite the significant progress that has been made in developing compounds for the treatment of cancer, there remains a need in the art to identify the particular structures and substitution patterns that form the compounds capable of modulating the function of particular protein kinases.
In a first aspect, the invention provides an indolinone compound having a structure set forth in formula (I): 
wherein:
(a) R4-R6, and R8-R10 are hydrogen;
(b) R1, R2, and R3 are each independently selected from the group consisting of hydrogen, halogen, carboxylic acid, optionally substituted ester, optionally substituted amide, optionally substituted alkyl, optionally substituted alkoxy, trihalomethyl, optionally substituted aryl, and optionally substituted heteroaryl; and
(c) R7 is selected from the group consisting of substituted alkyl, and substituted alkoxy;
or a pharmaceutically acceptable salt thereof.
Preferably,
(a) R1 is selected from the group consisting of hydrogen and optionally substituted alkyl, more preferably hydrogen;
(b) R2 and R3 are each independently selected from the group consisting of hydrogen, halo, carboxylic acid, optionally substituted heteroaryl, and optionally substituted phenyl. Preferably R2 is hydrogen, halo, phenyl, or carboxylic acid, more preferably hydrogen, phenyl, xe2x80x94COOH, chloro, fluoro or bromo. Preferably, R3 is hydrogen, halo, carboxylic acid, optionally substituted pyridyl, and phenyl optionally substituted with lower alkoxy or halo, more preferably phenyl, xe2x80x94COOH, pyridin-3-yl, 3-, or 4-methoxyphenyl or 4-fluorophenyl; and
(c) R7 is selected from the group consisting of lower alkyl substituted with a heteroaliphatic ring or dialkylamino; or lower alkoxy substituted with a heteroaliphatic ring or dialkylamino, preferably R7 is lower alkyl substituted with a heteroaliphatic ring or dialkylamino; more preferably R7 is 3-diethylaminopropyl or 3-pyrrolidin-1-yl-propyl, 2-dimethylaminoethoxy, 2-diethylaminoethoxy, 2-pyrrolidin-1-yl-ethoxy, or 2-morpholin-4-yl-ethoxy.
In a second aspect, the invention provides for an indolinone compound having a structure set forth in formula (II): 
wherein:
(a) R11-R14 are hydrogen;
(b) R15 and R16 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl, or R15 and R16 taken together with the nitrogen atom to which they are attached form a ring structure selected from the group consisting of a five-membered or six-membered heteroaromatic ring, a five-membered or six-membered heteroaliphatic ring, a nine-membered fused bicyclic heteroaromatic ring, and a ten-membered fused bicyclic heteroaromatic ring; and
(c) A is selected from the group consisting of formula (III), (IV), and (V): 
wherein:
(i) R19-R25 and R27-R31 are hydrogen;
(ii) R17 and R18 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted alkoxy provided that both R17 and R18 are not hydrogen; and
(iii) R26 is selected from the group consisting of optionally substituted alkyl;
or a pharmaceutically acceptable salt thereof.
In one preferred embodiment:
(i) R15 is hydrogen or alkyl, more preferably hydrogen or methyl;
(ii) R16 is hydrogen, alkyl, phenyl optionally substituted with one or two substituents selected from halo or unsubstituted lower alkyl or 5 or 6 membered heteroaryl; more preferably hydrogen, methyl, isopropyl, phenyl, pyridin-3-yl, 3-chlorophenyl, or 4-chloro-2-fluorophenyl; or
(iii) R15 and R16 together with the nitrogen to which they are attached form 2,3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoisoquinolin-2-yl ring wherein said rings are optionally substituted with halo or alkyl, preferably R15 and R16 together with the nitrogen atom to which they are attached form 2,3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, 5-bromo-2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoisoquinolin-2-yl;
(iv) A is group of formula III where:
R17 is hydrogen, methyl, or methoxy, preferably hydrogen; and
R18 is selected from the group consisting of lower alkoxy substituted with heteroalicyclic, preferably 2-pyrrolidin-1-yl-ethoxy and 2-morpholin-4-yl-ethoxy.
Another preferred group of compound is that wherein:
(i) R15 is hydrogen or alkyl, preferably hydrogen or methyl;
(ii) R16 is hydrogen, alkyl, phenyl optionally substituted with one or two substituents selected from halo or unsubstituted lower alkyl, or 5 or 6 membered heteroaryl; preferably hydrogen, methyl, isopropyl, phenyl, pyridin-3-yl, 3-chlorophenyl, or 4-chloro-2-fluorophenyl; or
R15 and R16 together with the nitrogen to which they are attached form 2,3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoisoquinolin-2-yl ring wherein said rings are optionally substituted with halo or alkyl, preferably 3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, 5-bromo-2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoisoquinolin-2-yl; and
(iii) A is group of formula IV where R26 is selected from the group consisting of optionally substituted alkyl, preferably hydrogen or methyl.
Another preferred group of compounds is that wherein:
(i) R15 is hydrogen or alkyl, preferably hydrogen or methyl;
(iii) R16 is hydrogen, alkyl, phenyl optionally substituted with one or two substituents selected from halo or unsubstituted lower alkyl or 5 or 6 membered heteroaryl; more preferably hydrogen, methyl, isopropyl, phenyl, pyridin-3-yl, 3-chlorophenyl, or 4-chloro-2-fluorophenyl; or R15 and R16 together with the nitrogen to which they are attached form 2,3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoquinolin-2-yl ring wherein said rings are optionally substituted with halo or alkyl, preferably R15 and R16 together with the nitrogen atom to which they are attached form 2,3-dihydroindol-1-yl, 2,3-dihydro-2H-quinolin-1-yl, 5-bromo-2,3-dihydro-2H-quinolin-1-yl, or 2,3-dihydro-2H-isoisoquinolin-2-yl;
(ii) A is group of formula V.
As used herein, the term xe2x80x9coptionally substituted alkylxe2x80x9d refers to an aliphatic hydrocarbon group. The alkyl moiety may be a xe2x80x9csaturated alkylxe2x80x9d group, which means that it does not contain any alkene or alkyne moieties. The alkyl moiety may also be an xe2x80x9cunsaturated alkylxe2x80x9d moiety, which means that it contains at least one alkene or alkyne moiety. An xe2x80x9calkenexe2x80x9d moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an xe2x80x9calkynexe2x80x9d moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, non-branched, or cyclic. Preferably, the alkyl group has 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as xe2x80x9c1 to 20xe2x80x9d refers to each integer in the given range; e.g., xe2x80x9c1 to 20 carbon atomsxe2x80x9d means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term xe2x80x9calkylxe2x80x9d where no numerical range is designated). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group is optionally substituted with one, two, or three substituents individually and independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, or halo. The term xe2x80x9csubstituted alkylxe2x80x9d means that the alkyl group as defined above carries at least one of the substituents listed above.
The term xe2x80x9coptionally substituted arylxe2x80x9d refer to an aromatic carbocyclic group of 6 to 12 ring atoms which has at least one ring having a conjugated xcfx80 electron system (e.g., phenyl, napthyl, tetrahydronaphthyl, and the like) which is optionally substituted with one, two, or three substituents independently selected from optionally susbstituted alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxyl amino, amido, nitro, and ester.
xe2x80x9cOptionally substituted phenylxe2x80x9d refers to a phenyl group that is optionally substituted with one, two, or three substituents independently selected from optionally susbstituted alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxyl amino, amido, nitro, and ester.
The term xe2x80x9ccycloalkylxe2x80x9d refers to a saturated, cyclic group of 3 to 6 carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) which is optionally substituted with one, two, or three substituents independently selected from optionally susbstituted alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxyl amino, amido, nitro, and ester.
The term xe2x80x9coptionally susbtituted heteroaryl or heteroaromatic ringxe2x80x9d refers to a ring system of 5 to 10 ring atoms in which one, two, three, or four of the atoms forming the backbone is a heteroatom selected from nitrogen, oxygen, or sulfur, the remaining being carbon e.g., pyridine, furan, pyrrole, indole, pyrazine, pyrimidine, tetrazole, imidazole, and the like. The heteroaromatic/heteroaryl ring may be single or fused bicyclic ring and wherein one of the rings may be partially or fully saturated e.g., 2,3-dihydroindole, 2,3-dihydroquinoline, 2,3-dihydroisoquinoline, and the like. The heteroaryl/heteroaromatic ring is optionally substituted with one, two, or three substituents independently selected from optionally susbstituted alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxyl amino, amido, nitro, and ester.
The term xe2x80x9coptionally susbtituted heteroaliphatic or heteroalicyclicxe2x80x9d refers to a saturated ring system of 5 to 9 ring atoms in which in which one, two, three, or four of the atoms forming the backbone is a heteroatom selected from nitrogen, oxygen, or sulfur, the remaining being carbon e.g., piperazine, piperidine, pyrrolidine, morpholine, tetrahydrofuran and the like. The heteroaliphatic ring is optionally substituted with one, two, or three substituents independently selected from optionally susbstituted alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxyl amino, amido, nitro, and ester.
The term xe2x80x9chalogenxe2x80x9d refers to an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine.
The term xe2x80x9ctrihalomethylxe2x80x9d refers to the xe2x80x94C(X)3 group, where X is a halogen group as defined above e.g., trifluoromethyl, trichloromethyl, tribromomethyl, and the like.
The term xe2x80x9coptionally substituted alkoxyxe2x80x9d is a group of formula xe2x80x94O-alkyl wherein alkyl is as defined above. The term xe2x80x9csubstituted alkoxyxe2x80x9d means that the alkyl group as defined above carries at least one of the substituents listed above. The term xe2x80x9calkoxyxe2x80x9d means that the alkyl chain in the alkoxy group as defined above is not substituted.
When X is hydrogen, then the alkoxy group becomes a xe2x80x9chydroxyxe2x80x9d group, i.e., xe2x80x94OH.
A xe2x80x9cnitroxe2x80x9d is a substituent of formula xe2x80x94NO2.
The term xe2x80x9calkylthioxe2x80x9d refers to xe2x80x94SR group where R is unsubstituted alkyl as defined above e.g., methylthio, ethylthio, and the like.
The term xe2x80x9caryloxyxe2x80x9d refers to xe2x80x94OR group where R is aryl group as defined above, e.g., phenoxy, and the like.
The term xe2x80x9carylthioxe2x80x9d refers to xe2x80x94SR group where R is aryl group as defined above e.g., phenylthio, and the like.
The term xe2x80x9cdialkylaminoxe2x80x9d means xe2x80x94NRR where each R is an unsubstituted alkyl group of 1-6 carbon atoms e.g., dimethylamino, diethylamino, and the like.
The term xe2x80x9coptionally substituted esterxe2x80x9d refers to xe2x80x94COOR where R is an alkyl group as defined above.
The term xe2x80x9coptionally substituted amidexe2x80x9d refers to xe2x80x94CONRaRb where Ra and Rb are hydrogen or unsubstituted lower alkyl.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, citric acid, maleic acid, succinic acid, tartaric acid, and the like.
Some of the preferred compounds of the invention that have the generic structure of formula I are listed in Table 1.
The above compounds have the structure of formula X, with the R101, R102, R103, and R112 substituents as defined in Table 2.
Some of the compounds of the invention that have the generic structure of formula II are listed in Table 3.
Some of the above compounds have the structure of formula XI, with the R104, R105, R106, and R113 substituents as defined in Table 4, while others have the structure of formula XII, with the R107, R108, and R109 substituents as defined in Table 5, and still others have the structure of formula XIII, with the R110 and R111 substituents as defined in Table 6.
In a further aspect, the invention relates to an indolinone compound having a structure set forth in formula VI: 
where
(a) R41 is selected from the group consisting of hydrogen, amide, and sulfonamide;
(b) R42 and R44 are each independently selected from the group consisting of hydrogen, halogen, and alkoxy;
(c) R43 is selected from the group consisting of hydrogen, alkyl, halogen, hydroxy, alkoxy, perhaloalkoxy, nitro, sulfone, and sulfonamide;
(d) R45 is selected from the group consisting of hydrogen, alkyl, nitro, and amide; and
(e) R46 is selected from the group consisting of hydrogen, alkyl, carboxylic acid, and amine.
Preferably, for the compounds of formula VI,
(a) R42 and R44 are each independently selected from the group consisting of hydrogen, chloro, and methoxy;
(b) R43 is selected from the group consisting of, hydrogen, fluoro, chloro, bromo, methoxy, hydroxy, methyl, ethyl, nitro, trifluoromethoxy, xe2x80x94SO2CH3, and xe2x80x94NHSO2CH3,
(c) R45 is selected from the group consisting of hydrogen, methyl, xe2x80x94NO2, and xe2x80x94NHC(O)C(CH3)3, and
(d) R46 is selected from the group consisting of hydrogen, methyl, xe2x80x94CH2N(CH3)2, xe2x80x94CH2N(CH2CH3)2, xe2x80x94CH2CH2C(O)OH, and xe2x80x94CH2CH2CH2C(O)OH.
The preferred compounds of the invention that have the structure set forth in formula VI are selected from the group consisting of 
where R41 is selected from the group consisting of xe2x80x94SO2N(X1)2, xe2x80x94NHSO2X1, xe2x80x94NHC(O)X1, where X1 is hydrogen or alkyl.
In another aspect, the invention relates to a combinatorial library of at least five indolinone compounds that can be formed by reacting an oxindole with an aldehyde where the oxindole has a structure set forth in formula VII or VIII: 
where
(a) R51-R54, R64, and R65 are hydrogen;
(b) R55 and R56 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl, or when taken together R55 and R56 form an optionally substituted five-membered or six-membered heteroaliphatic ring;
(c) R61-R63 are each independently selected from the group consisting of hydrogen, halogen, carboxylic acid, optionally substituted aryl, optionally substituted heteroaryl, and amide; and
where the aldehyde has a structure set forth in formula IX 
where
(d) R71 and R73-R75 are hydrogen;
(e) R72 is selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted alkoxy.
A xe2x80x9ccombinatorial libraryxe2x80x9d refers to all the compounds formed by the reaction of each compound of one dimension with a compound in each of the other dimensions in a multi-dimensional array of compounds. In the context of the present invention, the array is two dimensional and one dimension represents all the oxindoles of the invention and the second dimension represents all the aldehydes of the invention. Each oxindole may be reacted with each and every aldehyde in order to form an indolinone compound. All indolinone compounds formed in this way are within the scope of the present invention. Also within the scope of the present invention are smaller combinatorial libraries formed by the reaction of some of the oxindoles with all of the aldehydes, all of the oxindoles with some of the aldehydes, or some of the oxindoles with some of the aldehydes.
Preferably in the combinatorial library of the invention:
(a) R55 and R56 are each independently selected from the group consisting of hydrogen, methyl, isopropyl, 2-methoxyethyl, benzyl, 4-fluorobenzyl, 2-methoxyphenyl, 3-fluorophenyl, 4-fluorophenyl, 3-chlorophenyl, 3-pyridyl, or when taken together R55 and R56 form a ring selected from the group consisting of pyrrolidine, 4-methylpiperazine;
(b) R61-R63 are each independently selected from the group consisting of hydrogen, bromo, phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3-pyridyl, xe2x80x94COOH, xe2x80x94C(O)NHCH2CH3, xe2x80x94C(O)NHCH2C(O)OH, xe2x80x94C(O)NHCH(CH3)C(O)OH, xe2x80x94C(O)NHCH(CH(CH3)2)C(O)OH, and 
(c) R72 is selected from the group consisting of hydrogen 2-diethylamino-ethoxy, 3-diethylamino-1-yl-propyl, and 3-pyrrolidin-1-yl-propyl.
Even more preferably, in the combinatorial library of the invention the aldehyde is selected from the group consisting of 2-formyl-1H-indole, 2-formyl-5-(3-diethylamino-propyl)-1H-indole, 2-formyl-5-(2-dimethylaminoethoxy)-1H-indole, 2-formyl-5-(2-dimethylaminethoxy)-1H-indole, 2-formyl-5-(2-morpholin-4-yl-ethoxy)-1H-indole, 2-formyl-5-(2-pyrrolidine-1-yl-ethoxy)-1H-indole, 2-methyl-1H-indole-3-carbaldehy
and the oxindole is selected from the group consisting of 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid amide, 2methylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (4-chloro-2-fluoro-phenyl)-ethyl-amide, 5-(2,3-dihydro-indole-1-sulfonyl)-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (4-chloro-2-fluoro-phenyl)-methyl-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (4-chloro-2-fluoro-phenyl)-amide, 5-phenyl-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid [2-(4-methoxy-phenyl)-2-oxo-ethyl]-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-methoxy-phenyl)-amide, N-(2-oxo-2,3-dihydro-1H-indol-5-yl)-benzamide, cyclopentanecarboxylic acid (2-oxo-2,3-dihydro-1H-indol-5-yl)-amide, (2-oxo-2,3-dihydro-1H-indol-5-yl)-carbamic acid benzyl ester, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid-4-fluoro-benzylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid [3-(2-oxo-pyrrolidin-1-yl)-propyl]-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (1,2,3,4-tetrahydro-naphthalen-1-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (furan-2-ylmethyl)-amide, 3-(2-oxo-2,3-dihydro-1H-indole-5-sulfonylamino)-thiophene-2-carboxylic acid methyl ester, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chloro-phenyl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid m-tolylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (2-hydroxy-ethyl)-amide, 4-(2-oxo-2,3-dihydro-1H-indole-5-sulfonyl)-piperazine-1-carboxylic acid ethyl ester, 4-(2-oxo-2,3-dihydro-1H-indole-5-sulfonyl)-piperazine-1-carbaldehyde, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (2-methoxy-ethyl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide, 5-(4-methyl-piperazine-1-sulfonyl)-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid benzylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (2-methoxy-phenyl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid cyclopropylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid phenylamide, 5-(pyrrolidine-1-sulfonyl)-1,3-dihydro-indol-2-one, 5-(4-acetyl-piperazine-1-sulfonyl)-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid cyclohexylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (2-morpholin-4-yl-ethyl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid cyclobutylamide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (1-phenyl-ethyl)-amide, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid cyclopentylamide, 5-(2-pyrrolidin-1-yl-acetyl)-1,3-dihydro-indol-2-one, N-(2-oxo-2,3-dihydro-1H-indole-5-yl)-acetamide, (2-oxo-2,3-dihydro-1H-indole-5-yl)-carbamic acid tert-butyl ester, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid isopropylamide, toluene-4-sulfonic acid 2-(2-oxo-2,3-dihydro-1H-indole-4-yl)-ethyl ester, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid ethylamide, 4-phenyl-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (4-methoxy-phenyl)-amide, 4-(2-morpholin-4-yl-ethyl)-1,3-dihydro-indol-2-one, 4-(2-pyrrolidin-1-yl-ethyl)-1,3-dihydro-indol-2-one, 4-[2-(4-methyl-piperazin-1-yl)-ethyl]-1,3-dihydro indol-2-one, 4-[2-(3-bromo-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-(2-bromo-ethyl)-1,3-dihydro-indol-2-one, 4-(2-bromo-ethyl)-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (6-methoxy-4xe2x80x2-methylsulfanyl-biphenyl-3-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (4-methoxy-3-thiophen-3-yl-phenyl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid biphenyl-3-ylamide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (6-methoxy-3xe2x80x2-trifluoromethyl-biphenyl-3-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (4xe2x80x2-fluoro-6-methoxy-biphenyl-3-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (6-methoxy-[1,1xe2x80x2,4xe2x80x2,1xe2x80x3]terphenyl-3-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (6,4xe2x80x2-dimethoxy-biphenyl-3-yl)-amide, 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (3-chloro-4-methoxy-phenyl)-amide, isopropyl-carbamic acid-2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, 4-[2-(3-trifluoromethyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(5-chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(4-methoxy-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-(2-ethoxy-ethyl)-1,3-dihydro-indol-2-one, 4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one, ethyl-carbamic acid-2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, biphenyl-2-yl-carbamic acid-2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, 6-pyridin-3-yl-1,3-dihydro-indol-2-one, 6-phenyl-1 ,3-dihydro-indol-2-one, 6-(2-methoxy-phenyl)-1,3-dihydro-indol-2-one, 6-(3-methoxy-phenyl)-1,3-dihydro-indol-2-one, 6-(4-methoxy-phenyl)-1,3-dihydro-indol-2-one, and cyclohexyl-carbamic acid-2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester.
Some of the compounds of the invention that can be formed by the above combinatorial library are listed in Table 7.
Some of the above compounds have the structure of formula XIV, with the R114, R115, and R116 substituents as defined in Table 8, while others have the structure of formula XV, with the R107, R108, and R109 substituents as defined in Table 9.
In another aspect, the invention relates to a method of synthesizing a compound of formula I, II, or VI, which method comprises reacting an oxindole having the structure set forth in formula XV, XVI, or XVII, with an aldehyde or a ketone, having a structure set forth in formula XVIII, XIX, or XX. R1-R16, R4l-R46, and A in formulae XV-XX are as defined herein. 
The aldehyde of formula XX may be synthesized from the corresponding carboxylic acid or ester using the following reaction scheme. 
Rxe2x80x2 is hydrogen or alkyl, as defined herein.
To synthesize the compounds of the invention using the above methods a base may be used. The base is preferably a nitrogen base, an organic base, or an inorganic base. xe2x80x9cNitrogen basesxe2x80x9d are commonly used in the art and are selected from acyclic and cyclic amines. Examples of nitrogen bases include, but are not limited to, ammonia, methylamine, trimethylamine, triethylamine, aniline, 1,8-diazabicyclo[5.4.0]undec-7-ene, diisopropylethylamine, pyrrolidine, and piperidine. xe2x80x9cOrganic basesxe2x80x9d are bases that contain carbon atoms. Examples of organic bases include, but are not limited to, carbonate, bicarbonate, acetate, and formate anions. xe2x80x9cInorganic basesxe2x80x9d are bases that do not contain any carbon atoms. Examples of inorganic bases include, but are not limited to, hydroxide, phosphate, bisulfate, hydrosulfide, and amide anions. Those skilled in the art know which nitrogen base or inorganic base would match the requirements of the reaction conditions. In certain embodiments of the invention, the base used may be pyrrolidine or piperidine. In other embodiments the base may be the hydroxide anion, preferably used as its sodium or potassium salt.
The synthesis of the compounds of the invention takes place in a solvent. The solvent of the reaction is preferably a protic solvent or an aprotic solevent. xe2x80x9cProtic solventsxe2x80x9d are those that are capable of donating a proton to a solute. Examples of protic solvents include, but are not limited to, alcohols and water. xe2x80x9cAprotic solventsxe2x80x9d are those solvents that, under normal reaction conditions, do not donate a proton to a solute. Typical organic solvents, such as hexane, toluene, benzene, methylene chloride, dimethylformamide, dimethylsulfoxide, chloroform, tetrahydrofuran, are some of the examples of aprotic solvents. Other aprotic solvents are also within the scope of use by the present invention. In some preferred embodiments, the solvent of the reaction is an alcohol, which may preferably be isopropanol or most preferably ethanol. Water is another preferred protic solvent. Dimethylformamide, known in the chemistry art as DMF, and dimethylsulfoxide, known in the chemistry art as DMSO, are preferred aprotic solvents.
The synthetic method of the invention calls for the reaction to take place at elevated temperatures which are temperatures that are greater than room temperature. More preferably, the elevated temperature is preferably about 30-150xc2x0 C., more preferably is about 70-100xc2x0 C., and most preferably is about 70-90xc2x0 C., which is about the temperature at which ethanol boils (i.e., the boiling point of ethanol). By xe2x80x9caboutxe2x80x9d a certain temperature it is meant that the temperature range is preferably within 10xc2x0 C. of the listed temperature, more preferably within 5xc2x0 C. of the listed temperature, and most preferably within 2xc2x0 C. of the listed temperature. Therefore, by way of example, by xe2x80x9cabout 80xc2x0 C.xe2x80x9d it is meant that the temperature range is preferably 80xc2x110xc2x0 C., more preferably 80xc2x15xc2x0 C., and most preferably 80xc2x12 xc2x0 C.
The synthetic method of the invention may be accompanied by the step of screening a library for a compound of the desired activity and structurexe2x80x94thus, providing a method of synthesis of a compound by first screening for a compound having the desired properties and then chemically synthesizing that compound.
In another aspect, the invention features a pharmaceutical composition comprising (i) a physiologically acceptable carrier, diluent, or excipient; and (ii) an indolinone compound of the invention, as described herein. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The term xe2x80x9cpharmaceutical compositionxe2x80x9d refers to a mixture of an indolinone compound of the invention with other pharmaceutically acceptable diluents, excipients, or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions can be obtained by reacting compounds with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
The term xe2x80x9cpharmaceutically acceptablexe2x80x9d defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.
The term xe2x80x9ccarrierxe2x80x9d defines a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
The term xe2x80x9cdiluentxe2x80x9d defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
The invention also features a method of modulating the function of a PK or a protein phosphatase (PP) with an indolinone compound of the invention, comprising the step of contacting cells expressing the PK or a PP with the compound. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The term xe2x80x9cfunctionxe2x80x9d refers to the cellular role of PKs or PPs. These proteins include members that regulate many steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.
The term xe2x80x9ccatalytic activityxe2x80x9d, in the context of the invention, defines the rate at which a protein kinase phosphorylates a substrate or a protein phosphatase dephosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Phosphorylation or dephosphastase of a substrate occurs at the active-site of a protein kinase. The active-site is normally a cavity in which the substrate binds to the protein kinase or phosphatase and is connected to the product.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a molecule phosphorylated by a PK or dephosphorylated by a PP. The substrate is preferably a peptide and more preferably a protein.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the cellular function of a PK or PP. This function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
The term xe2x80x9cinhibitxe2x80x9d refers to decreasing the cellular function of a PK or PP. The PK or PP function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
The term xe2x80x9cmodulatesxe2x80x9d refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex forms between a protein kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the PK or PP and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the PK or PP and the natural binding partner depending on the concentration of the compound exposed to the PK or PP, and most preferably decreases the probability that a complex forms between the PK or PP and the natural binding partner. A modulator preferably activates the catalytic activity of a PK or PP, more preferably activates or inhibits the catalytic activity of a PK or PP depending on the concentration of the compound exposed to the PK or PP, or most preferably inhibits the catalytic activity of a PK or PP.
The term xe2x80x9ccomplexxe2x80x9d refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another.
The term xe2x80x9cnatural binding partnerxe2x80x9d refers to polypeptides that bind to a PK or PP in cells. Natural binding partners can play a role in propagating a signal in a PK or PP signal transduction process. A change in the interaction between a PK or PP and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the PK or PP/natural binding partner complex.
A PK or PP natural binding partner can bind to a PK""s or PP""s intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of 10xe2x88x926 M or less. In addition, a natural binding partner can also transiently interact with a protein kinase intracellular region and chemically modify it. PK or PP natural binding partners are chosen from a group that includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods of determining changes in interactions between PK or PP and their natural binding partners are readily available in the art.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to mixing a solution comprising an indolinone compound of the invention with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethylsulfoxide (DMSO), which facilitates the uptake of the indolinone compound or compounds into the cells of the methods. The solution comprising the indolinone compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
The indolinone compounds of the invention preferably modulate the activity of the PK or PP in vitro. These compounds preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in the Examples below). It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The invention also features a method of identifying indolinone compounds that modulate the function of PK or PP, comprising the following steps: (a) contacting cells expressing the PK or PP with the compound; and (b) monitoring an effect upon the cells. The effect upon the cells is preferably a change or an absence of a change in cell phenotype, more preferably it is a change or an absence of a change in cell proliferation, even more preferably it is a change or absence of a change in the catalytic activity of the PK or PP, and most preferably it is a change or absence of a change in the interaction between the PK or PP with a natural binding partner, as described herein. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The term xe2x80x9cmonitoringxe2x80x9d refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in cell phenotype, cell proliferation, PK or PP catalytic activity, or in the interaction between a PK or PP and a natural binding partner.
The term xe2x80x9ceffectxe2x80x9d describes a change or an absence of a change in cell phenotype or cell proliferation. xe2x80x9cEffectxe2x80x9d can also describe a change or an absence of a change in the catalytic activity of the pro PK or PP. xe2x80x9cEffectxe2x80x9d can also describe a change or an absence of a change in an interaction between the PK or PP and a natural binding partner.
The term xe2x80x9ccell phenotypexe2x80x9d refers to the outward appearance of a cell or tissue or the function of the cell or tissue. Examples of cell phenotype are cell size (reduction or enlargement), cell proliferation (increased or decreased numbers of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death), or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art.
In a preferred embodiment, the invention features a method for identifying other compounds capable of modulating the activity of PK or PP using the indolinones of the invention as a reference which method comprises the following steps: (a) lysing the cells to render a lysate comprising PK or PP; (b) adsorbing the PK or PP to an antibody; (c) incubating the adsorbed PK or PP with a substrate or substrates in the presence or absence of test compound or an indolinone compound of the present invention; and (d) adsorbing the substrate or substrates to a solid support or antibody the effect upon the cells is then monitored and the step of monitoring the effect on the cells comprises measuring the phosphate concentration of the substrate or substrates. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The term xe2x80x9cantibodyxe2x80x9d refers to an antibody (e.g., a monoclonal or polyclonal antibody), or antibody fragment, having specific binding affinity to PK or PP or its fragment.
By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to target (PK or PP) polypeptides with greater affinity than it binds to other polypeptides under specified conditions. Antibodies having specific binding affinity to a PK or PP may be used in methods for detecting the presence and/or amount of a PK or PP in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the PK or PP. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
In yet another aspect, the invention features a method for treating a disease related to unregulated tyrosine kinase or phosphatase signal transduction, where the method includes the step of administering to a subject in need thereof a therapeutically effective amount of an indolinone compound as described herein. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
The term xe2x80x9ctreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d does not necessarily mean total cure. Any alleviation of any undesired symptom of the disease to any extent or the slowing down of the progress of the disease can be considered treatment. Furthermore, treatment may include acts which may worsen the patient""s overall feeling of well being or appearance. For example, the administration of chemotherapy in cancer patients which may leave the patients feeling xe2x80x9csickerxe2x80x9d is still considered treatment.
The invention also features a method of regulating tyrosine kinase or phosphatase signal transduction comprising administering to a subject a therapeutically effective amount of an indolinone compound as described herein. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention.
Furthermore, the invention features a method of preventing or treating an abnormal condition in an organism, where the abnormal condition is associated with an aberration in a signal transduction pathway characterized by an interaction between a PK or PP and a natural binding partner, where the method comprises the following steps: (a) administering an indolinone compound as described herein; and (b) promoting or disrupting the abnormal interaction. The organism is preferably a mammal and the abnormal condition is preferably cancer. It is understood that the indolinone compound of the invention may be one of formula I, II, or VI, or a compound obtained from the combinatorial library of the invention. The abnormal condition may also preferably be selected from the group consisting of hypertension, depression, generalized anxiety disorder, phobias, post-traumatic stress syndrome, avoidant personality disorder, sexual dysfunction, eating disorders, obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer""s disease, obsessive-compulsive disorder, panic disorder, memory disorders, Parkinson""s disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal tract disorders.
The term xe2x80x9caberrationxe2x80x9d, in conjunction with a signal transduction process, refers to a PK or PP that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type PK or PP activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
The term xe2x80x9cpromoting or disrupting the abnormal interactionxe2x80x9d refers to a method that can be accomplished by administering a compound of the invention to cells or tissues in an organism. A compound can promote an interaction between a PK or PP and natural binding partners by, for example, forming favorable interactions with multiple atoms at the complex interface. Alternatively, a compound can inhibit an interaction between a PK or PP and natural binding partners by compromising favorable interactions formed between atoms at the complex interface.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
Utility
The present invention relates to compounds capable of regulating and/or modulating PK or PP signal transduction and more particularly receptor and non-receptor tyrosine kinase signal transduction.
Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects to the extracellular microenvironment). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.
It has been shown that tyrosine phosphorylation sites in growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423; Songyang et al, 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with receptor tyrosine kinases have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
Tyrosine kinase signal transduction results in, among other responses, cell proliferation, differentiation and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis).
This invention is therefore directed to compounds which regulate, modulate and/or inhibit tyrosine kinase signal transduction by affecting the enzymatic activity of the RTKs and/or the non-receptor tyrosine kinases and interfering with the signal transduced by such proteins. More particularly, the present invention is directed to compounds which regulate, modulate and/or inhibit the RTK and/or non-receptor tyrosine kinase mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinoma, sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers. The present invention is also directed to compounds used in a therapeutic approach to cure non-solid tumor cancers, such as, leukemia.
I. Target Diseases to be Treated by the Compounds of the Invention
The compounds described herein are useful for treating disorders related to unregulated tyrosine kinase signal transduction, including cell proliferative disorders, fibrotic disorders and metabolic disorders.
Cell proliferative disorders which can be treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders.
Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness, and Von Hippel-Lindau (VHL) disease, which is an inherited multi-system disorder characterized by abnormal growth of blood vessels in certain parts of the body. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated.
Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis (see, below).
Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47S-54S.
PKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like the EGFR (Tuzi et al., 1991, Br. J Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al, 1989, Science 244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene 7:627-633) are overexpressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor overexpressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol. 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, the EGFR receptor has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer. The PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and prostate cancer. The RTK c-met has been generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, c-met has been linked to malignant tumor formation. More specifically, the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression of the c-met gene has been detected in patients with Hodgkin""s disease, Burkitt""s disease, and the lymphoma cell line.
The IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g., human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res. 50:2511-2517). In addition, IGF-I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The importance of the IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Gene Expression 1:301-326. In a series of recent publications, Baserga even suggests that IGF-I-R plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res. 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol. 14:4588-4595.
The association between abnormalities in RTKs and disease are not restricted to cancer, however. For example, RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. These diseases include, but are not limited to hypertension, depression, generalized anxiety disorder, phobias, post-traumatic stress syndrome, avoidant personality disorder, sexual dysfunction, eating disorders, obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer""s disease, obsessive-compulsive disorder, panic disorder, memory disorders, Parkinson""s disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal tract disorders. For example, the EGF-R is indicated in corneal and dermal wound healing. Defects in the Insulin-R and the IGF-IR are indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., 1994, DNandP 7:334-339.
Not only receptor type tyrosine kinases, but also many cellular tyrosine kinases (CTKs) including src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al., 1992, FASEB J. 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus in indications of the present invention. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60c-src transmits oncogenic signals of many receptors. For example, overexpression of EGF-R or HER2/neu in tumors leads to the constitutive activation of pp60c-src, which is characteristic for the malignant cell but absent from the normal cell. On the other hand, mice deficient for the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders. Similarly, Zap-70 is implicated in T-cell signaling.
Furthermore, the identification of CTK modulating compounds to augment or even synergize with RTK aimed blockers is an aspect of the present invention.
Finally, both RTKs and non-receptor type kinases have been connected to hyperimmune disorders.
The compounds of the present invention are also effective in treating diseases that are related to the PYK-2 protein. This protein, its cellular function, and diseases related to them are set forth in detail in U.S. Pat. No. 5,837,524, issued Nov. 17, 1998, by Lev et al., and entitled xe2x80x9cPYK2 RELATED PRODUCTS AND METHODSxe2x80x9d (Lyon and Lyon Docket No. 209/070), U.S. Pat. No. 5,837,815, issued Nov. 17, 1998, by Lev et al., and entitled xe2x80x9cPYK2 RELATED PRODUCTS AND METHODSxe2x80x9d (Lyon and Lyon Docket No. 211/121), U.S. patent application Ser. No. 08/987,689, filed Dec. 9, 1997, by Lev et al., and entitled xe2x80x9cPYK2 RELATED PRODUCTS AND METHODSxe2x80x9d (Lyon and Lyon Docket No. 230/110), U.S. patent application Ser. No. 09/165,062, filed Oct. 1, 1998, by Lev et al., and entitled xe2x80x9cPYK2 RELATED PRODUCTS AND METHODSxe2x80x9d (Lyon and Lyon Docket No. 237/201), International Publication Number WO98/26054, published Jun. 18, 1998, by Lev et al., and entitled xe2x80x9cPYK2 RELATED PRODUCTS AND METHODSxe2x80x9d (Lyon and Lyon Docket No. 222/126), and International Application Number US 98/27871, filed Dec. 31, 1998, by Schlessinger et al., and entitled xe2x80x9cPYK2 AND INFLAMMATIONxe2x80x9d (Lyon and Lyon Docket No. 236/075), all of which are hereby incorporated by reference herein in their entirety, including any drawings.
In addition, some of the compounds of the present invention are effective against rhematoid arthritis (RA). RA is a chronic inflammatory disease mediated by multiple cell types and cellular processes. Included in these are the infiltration of macrophages and T cells, and the involvement of angiogenesis. The utility of small molecule inhibitors for the treatment of RA was investigated in a rat collagen induced arthritis model. Some of the compounds of the present invention inhibit the tyrosine kinases Flk-1/KDR, pyk2, and ZAP-70 to varying degrees in biochemical kinase assays. In addition, the compounds are active in cellular assays targeted to cells implicated in the pathogenesis of RA: inhibition of T cell proliferation mediated by ZAP-70 activity, inhibition of VEGF stimulated HUVEC proliferation mediated by Flk-1/KDR activation, and inhibition of TNF-xcex1 production from murine bone marrow derived macrophages mediated by pyk2 activation. Finally, in arodent collagen induced arthritis model, which mimics the histological and pathological changes associated with human RA, some of the compounds of the invention are efficacious in inhibiting joint swelling when dosed daily from the time of collagen immunization.
II. The KDR/FLK-1 Receptor and VEGF
Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy. Folkman and Shing, 1992, J. Biological Chem. 267:10931-34. However, many diseases are driven by persistent unregulated or inappropriate angiogenesis. For example, in arthritis, new capillary blood vessels invade the joint and destroy the cartilage. In diabetes, new capillaries in the retina invade the vitreous, bleed and cause blindness. Folkman, 1987, in: Congress of Thrombosis and Haemostasis (Verstraete, et. al, eds.), Leuven University Press, Leuven, pp. 583-596. Ocular neovascularization is the most common cause of blindness and dominates approximately twenty (20) eye diseases.
Moreover, vasculogenesis and/or angiogenesis have been associated with the growth of malignant solid tumors and metastasis. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow. Furthermore, the new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and to metastasize to distant sites in the body. Folkman, 1990, J. Natl. Cancer Inst. 82:4-6; Klagsbrunn and Soker, 1993, Current Biology 3:699-702; Folkman, 1991, J. Natl., Cancer Inst. 82:4-6; Weidner et al., 1991, New Engl. J. Med. 324:1-5.
Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (aFGF, bFGF), vascular endothelial growth factor (VEGF) and placental growth factor. Unlike aFGF and bFGF, VEGF has recently been reported to be an endothelial cell specific mitogen. Ferrara and Henzel, 1989, Biochem. Biophys. Res. Comm. 161:851-858; Vaisman et al, 1990, J. Biol. Chem. 265:19461-19566.
Thus, the identification of the specific receptors to which VEGF binds is an important advancement in the understanding of the regulation of endothelial cell proliferation. Two structurally closely related RTKs have been identified to bind VEGF with high affinity: the fit-i receptor (Shibuya et al., 1990, Oncogene 5:519-524; De Vries et al., 1992, Science 255:989-991) and the KDR/FLK-1 receptor, (also referred to as VEGFR2) discussed in the U.S. patent application Ser. Nos. 08/193,829 and 08/965,598. Consequently, it had been surmised that these RTKs may have a role in the modulation and regulation of endothelial cell proliferation.
Evidence, such as the disclosure set forth in copending U.S. application Ser. Nos. 08/193,829 and 08/965,598, strongly suggests that VEGF is not only responsible for endothelial cell proliferation, but also is a prime regulator of normal and pathological angiogenesis. See generally, Klagsbum and Soker, 1993, Current Biology 3:699-702; Houck et al., 1992, J. Biol. Chem. 267:26031-26037. Moreover, it has been shown that KDR/FLK-1 and fit-1 are abundantly expressed in the proliferating endothelial cells of a growing tumor, but not in the surrounding quiescent endothelial cells. Plate et al., 1992, Nature 359:845-848; Shweiki et al., 1992, Nature 359:843-845.
III. Identification of Agonists and Antagonists to the KDR/FLK-1 Receptor
In view of the deduced importance of RTKs in the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis, many attempts have been made to identify RTK xe2x80x9cinhibitorsxe2x80x9d using a variety of approaches. These include the use of mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors and antibodies (International Publication No. WO 94/10202; Kendall and Thomas, 1994, Proc. Natl. Acad. Sci. USA 90:10705-10709; Kim et al., 1993, Nature 362:841-844); and RNA ligands (Jellinek et al., 1994, Biochemistry 33:10450-10456).
Furthermore, tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani et al., 1994, Proc. Am. Assoc. Cancer Res. 35:2268), and inhibitors acting on receptor tyrosine kinase signal transduction pathways, such as protein kinase C inhibitors have been identified (Schuchter et al., 1991, Cancer Res. 51:682-687); Takano et al., 1993, Mol. Bio. Cell 4:358A; Kinsella et al., 1992, Exp. Cell Res. 199:56-62; Wright et al., 1992, J. Cellular Phys. 152:448-57).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer.
Consequently, there is an unmet need for the identification and generation of effective small compounds which selectively inhibit the signal transduction of the KDR/FLK-1 receptor in order to effectively and specifically suppress vasculogenesis.
Some of the compounds of the present invention demonstrate excellent activity in biological assays and thus these compounds and related compounds are expected to be effective in treating Flk related disorders such as those driven by persistent unregulated or inappropriate angiogenesis.
Pharmaceutical Formulations and Routes of Administration
The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in xe2x80x9cRemington""s Pharmaceutical Sciences,xe2x80x9d Mack Publishing Co., Easton, Pa., latest edition.
1. Routes of Administration
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
2. Composition/Formulation
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80(trademark), and 65% w/v polyethylene glycol-300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80(trademark); the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the PK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
3. Effective Dosage
Pharmaceutical compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity). Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition. (See e.g., Fingl et al., 1975, in xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d, Ch. 1 p.1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject""s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
4. Packaging
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the polynucleotide for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
IV. Biological Activity of the Indolinone Compounds of the Invention
Some of the indolinone compounds of the present invention were tested for their ability to inhibit most of protein tyrosine kinase activity. The biological assays and results of these inhibition studies are reported herein. The methods used to measure indolinone compound modulation of protein kinase function are similar to those described in International Publication No. WO 98/07695, published Mar. 26, 1998, by Tang et al., and entitled xe2x80x9cIndolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease,xe2x80x9d with respect to the high throughput aspect of the method. The WO 98/07695 publication is incorporated herein by reference in its entirety, including any drawings.
V. Pharmaceutical Compositions and Administration of Indolinone Compounds of the Invention
Methods of preparing pharmaceutical formulations of the compounds, methods of determining the amounts of compounds to be administered to a patient, and modes of administering compounds to an organism are disclosed in International Publication No. WO 98/07695, by Tang et al., and entitled xe2x80x9cIndolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease,xe2x80x9d and International Publication No. WO 96/22976, by Buzzetti et al., and entitled xe2x80x9cHydrosoluble 3-Arylidene-2-Oxindole Derivatives as Tyrosine Kinase Inhibitors,xe2x80x9d published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.